Many different techniques have been developed in the field of fluorescence microscopy to restrict excitation light to a thin region of a specimen in order to improve the signal-to-background noise ratio and the spatial resolution of specimen features or components of interest. Total internal reflection fluorescence (“TIRE”) microscopy is a technique in which a beam of excitation light is restricted to a thin region of the specimen adjacent to the interface between the specimen and a transparent substrate, such as a slide, coverslip or dish. The excitation light is transmitted into the substrate and strikes the interface at a nonzero angle of incidence. When the angle of incidence is larger than a critical angle with respect to the interface normal, the light experiences total internal reflection. In other words, if the refractive index of the specimen is lower than that of the substrate and the angle of incidence is greater than the critical angle, no excitation light can pass into the specimen and the excitation light is reflected back into the substrate. But, the reflected light generates an electromagnetic field that penetrates beyond the interface into the specimen as an evanescent wave with the same wavelength as the excitation light that excites fluorescence within a thin region of the specimen near the interface. Objective-based TIRF microscopes direct the excitation light along the outer edge of the objective lenses so that light exits the objective and strikes the interface with an angle of incidence greater than the critical angle. These instruments, which use oil-immersion objectives with a high numerical aperture, are increasing in popularity because they can be used to image live cell specimens in coverslip-bottom dishes.
On the other hand, objective-based TIRF microscopes present several challenges when dealing with multiple wavelength TIRF. For instance, multiple wavelength TIRF microscopes use a multiple wavelength excitation beam directed along the outer edge of the objective lenses, but the outer edges of the lenses cause chromatic aberrations in the beam. One technique used to account for chromatic aberrations is to switch between the different excitation wavelengths by mechanically steering and refocusing the multiple wavelength beam so that a selected wavelength strikes the interface with an angle of incidence greater than the critical angle. However, steering and refocusing the beam for each wavelength takes time, prevents simultaneous imaging with more than one wavelength, and requires additional mechanical systems to change the position of the excitation beam source, which increases the cost of an objective-based TIRF microscope. For the above described reasons, engineers, scientists, and fluorescent microscope manufacturers continue to seek fast, efficient, and cost effective systems that correct for chromatic aberrations in multiple wavelength TIRF microscopy.